The present technology relates to an optical measuring apparatus that optically detects a sample using a flow path. More particularly, the present disclosure relates to an optical measuring apparatus that optically detects a sample flowing through a flow path, a flow cytometer using the optical measuring apparatus, and an optical measuring method.
In recent years, with the development of analysis techniques, a technique of causing biomicroparticles such as cells or microorganisms or microparticles such as microbeads to flow through a flow path, individually measuring the microparticles in the flow process, analyzing the measured microparticles, and then sorting a desired microparticle has been developed. As a representative example of the technique of analyzing and sorting microparticles using a flow path, an analysis technique called flow cytometry has been rapidly improving.
Flow cytometry refers to an analysis technique in which microparticles are analyzed and sorted such that microparticles of an analysis target flow in a fluid in an aligned state, the microparticles are irradiated with a laser beam, and fluorescent light or scattered light emitted from each microparticle is detected. Processes of flow cytometry are roughly classified into (1) a flow system, (2) an optical system, (3) an electrical analysis system, and (4) a sorting system as follows.
(1) Flow System
In the flow system, microparticles of an analysis target are aligned in a flow cell (flow path). More specifically, a sheath fluid flows into a flow cell at a predetermined speed, and a sample fluid including microparticles slowly flows into a central core of the flow cell in this state. At this time, the fluids do not mix with each other due to the principle of laminar flow, and flow (laminar flow) in which layers form is formed. Inflows of the sheath fluid and the sample fluid are adjusted according to the size of the microparticles of an analysis target or the like, and then the sheath fluid and the sample fluid are caused to flow through in a state in which respective microparticles are aligned.
(2) Optical System
In the optical system, the microparticles of the analysis target are irradiated with light such as a laser, and fluorescent light or scattered light emitted from the microparticles is detected. The flow system (1) causes the microparticles to flow through a laser irradiating unit in a state in which the respective microparticles are aligned. Then, each time each microparticle passes through, fluorescent light or scattered light emitted from the microparticle is detected for each parameter using an optical detector, and a characteristic of each microparticle is analyzed.
(3) Electrical Analysis System
In the electrical analysis system, optical information detected by the optical system is converted into an electric signal (voltage pulse). The converted electric signal is subjected to analog-to-digital (AD) conversion, and a histogram is extracted based on the data through an analysis computer and software, and then analysis is performed.
(4) Sorting System
In the sorting system, the measured microparticles are separated and collected. As a representative sorting technique, there is a technique in which sorting is performed such that positive or negative charges are applied to the measured microparticles, the flow cell is interposed between two deflecting plates having a potential difference therebetween, and so the charged microparticles are attracted to either one of the deflecting plates according to the charges thereof.
Techniques of analyzing and storing the microparticles in the flow path such as flow cytometry have been widely used in various kinds of fields such as the medical field, the drug discovery field, the clinical examination field, the food field, the agricultural field, the engineering field, the forensic medicine field, and the criminal identification field. Particularly, in the medical field, it has undertaken an important role in pathology, tumor immunology, transplantation, genetics, regenerative medicine, chemotherapy, and the like.
The technique of analyzing and storing the microparticles in the flow path is necessary in a very broad range of fields as described above, and techniques related to the processes of (1) to (4) are being developed from day to day. For example, as a technique related to the optical system of (2), Japanese Patent Application Laid-Open (JP-A) No. 2009-063305 discloses a technique in which position information of a sample in a flow path is acquired by irradiating a sample with directional light, and directional light is irradiated based on the position information, so that non-uniform irradiation and deviation of an irradiation position or a focus position are prevented.
Further, Japanese Patent Application Laid-Open No. 2010-256278 discloses a technique in which optical axis alignment is performed such that a detection flow path is positioned with respect to an optical axis of excitation light by controlling movement of a stage with a microorganism examination chip therein based on a quantity of fluorescent light detected by a first detector that detects fluorescent light emitted from a microorganism flowing in the detection flow path and converts the detected fluorescent light into an electric signal.
Meanwhile, optical axis alignment is important in terms of improvement in accuracy to detect a sample flowing through a flow path. Particularly, in recent years, a technique of detecting a cell using a microscale flow path installed in a chip has been developed and put into practical use, and in this lab-on-chip technique, a disposable chip is frequently used. For this reason, how efficiently optical axis alignment is performed each time is very important in terms of improvement in experimental efficiency.
In the related art, when a sample flowing through a flow path is detected, optical axis alignment is performed by observation with an external oscilloscope or while viewing the amplitude of a digital raw waveform or a data plot. In this case, the amplitude of a waveform can be intuitively determined by viewing the waveform, but the frequency of a sample flowing through the flow path can be determined by reading, for example, numerical data (event/sec) of a waveform graph, and optical axis adjustment has to be performed according to the numerical value.
Further, it is difficult to cause samples to flow through in a state in which the respective samples are aligned depending on a degree of the density of samples flowing through the flow path, and when two or more samples flow through in a dense state, an abort rate in which obtained waveform data has two or more peaks may occur. In preventing the abort rate, it is very important to read the frequency of sample flowing through the flow path.